Design of customizable long linear DNA substrates with controlled end modifications for single-molecule studies

2020 ◽  
Vol 592 ◽  
pp. 113541 ◽  
Author(s):  
Stefan H. Mueller ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen ◽  
Jacob S. Lewis
Keyword(s):  
Single Molecule ◽  
Linear Dna ◽  
Single Molecule Studies ◽  
Dna Substrates

scholarly journals High-yield purification of exceptional-quality, single-molecule DNA substrates

2021 ◽  
Vol 8 (1) ◽  
pp. e145
Author(s):  
Yue Lu ◽  
Piero Bianco
Keyword(s):  
Single Molecule ◽  
Exchange Resin ◽  
High Yield ◽  
The Past ◽  
Magnetic Tweezer ◽  
Single Molecule Studies ◽  
Purification Methods ◽  
Dna Substrates ◽  
High Yields ◽  
Dna Substrate

Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.

scholarly journals Efficient modification of λ-DNA substrates for single-molecule studies

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Yoori Kim ◽  
Armando de la Torre ◽  
Andrew A. Leal ◽  
Ilya J. Finkelstein
Keyword(s):  
Single Molecule ◽  
Single Molecule Studies ◽  
Dna Substrates

Biology on track: single-molecule visualisation of protein dynamics on linear DNA substrates

2020 ◽  
Author(s):  
Gurleen Kaur ◽  
Lisanne M. Spenkelink
Keyword(s):  
Single Molecule ◽  
Molecular Motor ◽  
Imaging Techniques ◽  
Biological Research ◽  
Dna Interactions ◽  
Single Molecule Fluorescence ◽  
Linear Dna ◽  
Protein Dna Interactions ◽  
Dna Substrates

Abstract Single-molecule fluorescence imaging techniques have become important tools in biological research to gain mechanistic insights into cellular processes. These tools provide unique access to the dynamic and stochastic behaviour of biomolecules. Single-molecule tools are ideally suited to study protein–DNA interactions in reactions reconstituted from purified proteins. The use of linear DNA substrates allows for the study of protein–DNA interactions with observation of the movement and behaviour of DNA-translocating proteins over long distances. Single-molecule studies using long linear DNA substrates have revealed unanticipated insights on the dynamics of multi-protein systems. In this review, we provide an overview of recent methodological advances, including the construction of linear DNA substrates. We highlight the versatility of these substrates by describing their application in different single-molecule fluorescence techniques, with a focus on in vitro reconstituted systems. We discuss insights from key experiments on DNA curtains, DNA-based molecular motor proteins, and multi-protein systems acting on DNA that relied on the use of long linear substrates and single-molecule visualisation. The quality and customisability of linear DNA substrates now allows the insertion of modifications, such as nucleosomes, to create conditions mimicking physiologically relevant crowding and complexity. Furthermore, the current technologies will allow future studies on the real-time visualisation of the interfaces between DNA maintenance processes such as replication and transcription.

scholarly journals Multi-site internal modification of long DNA substrates for single-molecule studies

2016 ◽  
Author(s):  
Armando de la Torre ◽  
Yoori Kim ◽  
Andrew A. Leal ◽  
Ilya J. Finkelstein
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Nuclear Antigen ◽  
Triplet Repeat ◽  
Chemical Modifications ◽  
Site Specific ◽  
Specific Modification ◽  
Modified Dna ◽  
Single Molecule Studies ◽  
Dna Substrates

AbstractSingle-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. DNA isolated from bacteriophage λ (λ-DNA) is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific DNA sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into λ-DNA with 90-100% yield. Furthermore, various DNA structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of Proliferating Cell Nuclear Antigen (PCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that PCNA can load on both 5’-ssDNA flaps and a 13xCAG triplet repeat. However, PCNA remains trapped on the 13xCAG structure, confirming a proposed mechanism for triplet repeat expansion. Although we focus on λ-DNA, this method is applicable to all long DNA substrates. We anticipate that this molecular toolbox will be broadly useful for both ensemble‐ and single-molecule studies that require site-specific modification of long DNA substrates.

scholarly journals Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Facilitated Diffusion ◽  
One Dimensional ◽  
Bacterial Transcription ◽  
Fluorescence Measurements ◽  
Dimensional Diffusion ◽  
Single Molecule Studies ◽  
Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

Breaking Down the Supramolecular Ensemble: Single-Molecule Studies of the Concentration Dependence of Main-Chain Supramolecular Polymer Molecular Weight Distributions on Surfaces

2010 ◽  
Vol 63 (4) ◽  
pp. 624
Author(s):  
Michael J. Serpe ◽  
Jason R. Whitehead ◽  
Stephen L. Craig
Keyword(s):  
Molecular Weight ◽  
Single Molecule ◽  
Monomer Concentration ◽  
Supramolecular Polymer ◽  
Force Microscopy ◽  
Molecular Weight Distributions ◽  
Atomic Force ◽  
Multi Stage ◽  
Single Molecule Studies ◽  
Supramolecular Ensemble

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.

scholarly journals Single-Molecule Studies of the Na+/K+-ATPase

2011 ◽  
Vol 100 (3) ◽  
pp. 464a
Author(s):  
Promod R. Pratap ◽  
Gregor Heiss ◽  
Martin Sikor ◽  
Don C. Lamb ◽  
Max Burnett
Keyword(s):  
Single Molecule ◽  
Single Molecule Studies
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